The blood-brain barrier is a complex dynamic interface that transduces biomechanical and biochemical signals from the vascular system and the brain and is responsible for maintaining homeostasis of the brain by regulating exchange of water, ions, nutrients, metabolites, neurotransmitters, and other cells (e.g., leukocytes), while limiting entry of potentially toxic xenobiotics in the blood (Abbott et al., 2010; Hawkins and Davis, 2005; Begley and Brightman, 2003).
The BBB is formed, in part, by highly specialized endothelial cells that line brain capillaries. The tight junctions formed by brain microvascular endothelial cells (BMECs) regulate paracellular transport, whereas transcellular transport is regulated by specialized transporters, pumps, and receptors (Abbott et al., 2010; Ohtsuki and Terasaki, 2007; Ueno, 2009; Hartz and Bauer, 2011; Hawkins et al., 2002; Chishty et al., 2001; Demeule et al., 2002). This barrier regulates transport by transducing signals from the vascular system and the central nervous system. Mounting evidence indicates that the structure and function of the BBB is controlled by the complex interplay between BMECs, astrocytes, pericytes, basement membrane proteins, components of the blood, and the shear stress associated with blood flow.
In 1980, it was shown that co-culture of brain capillary endothelial cells and astrocytes was essential to maintaining several important features of the BBB (Debault and Cancilla, 1980). Strong evidence indicates that astrocytes upregulate many BBB features leading to the formation of improved tight junctions, the expression and polarized localization of transporters to the apical or basal membranes, and specialized enzyme systems (Debault and Cancilla, 1980; Janzer and Raff, 1987; Abbott, 2002; Abbott et al., 2006; Haseloff et al., 2005).
The lack of a physiologically relevant in vitro model system, however, has been identified as a significant barrier to progress in this field. (Neuwelt et al., 2011; Cecchelli et al., 2007; Neuwelt et al., 2008). Current models of the BBB typically are two-dimensional (2D) co-culture models with cells plated on opposite sides of a porous polymer membrane or hollow cylinder. Generally, in vitro 2D models are based on a monolayer of BMECs plated on a porous membrane located between two chambers. Transendothelial resistance (TEER) measurements or permeability measurements are used to assess barrier properties. A wide range of configurations have been studied including: 2D monolayers of BMECs and astrocytes plated on opposite sides of the membrane support, BMECs and pericytes plated on opposite sides of a membrane support with astrocytes or astrocyte extract in one chamber, and variations where the BMECs are plated on membranes coated with ECM or basement membrane proteins (Hartmann et al., 2007; Cucullo et al., 2011; Nakagawa et al., 2009; Zozulya et al., 2008; Weidenfeller et al., 2007; Tilling et al., 1998; Rubin et al., 1991; Bickel, 2005; Ma et al., 2005; Siddharthan et al., 2007; Lundquist and Renftel, 2002; Lundquist et al., 2002).
The 2D models known in the art are not sufficiently close to capturing the physical and biological characteristics of the BBB to be widely used for BBB research. Key limitations of these models include no paracrine signaling between cells, astrocytes not in a physiologically relevant 3D matrix, and no shear flow.
Surprisingly little research aimed at developing an artificial capillary platform has been done, to date. Tien and coworkers have reported on an artificial vessel formed by seeding vascular endothelial cells on the internal surface of a cylindrical channel in a collagen matrix (Poller et al., 2008; Weksler et al., 2005; Hawkins et al, 2005; Wong et al., 2010). This vessel, however, is not a model for the BBB and does not include the relevant cell types.
The most advanced model of an artificial brain capillary has been developed by Janigro and co-workers and involves co-culture of endothelial cells and astrocytes onto impermeable, hollow cylinder polypropylene fibers (FIG. 1; Stanness et al., 1997; Cucullo et al., 2007). The polypropylene fibers have an inner diameter of 330 μm, a wall thickness of 150 μm, and 500-nm diameter pores in the wall. Bundles of these fibers are encased in cartridges with an external perfusion circuit, maintaining flow of culture medium with CO2. Brain capillary endothelial cells and astrocytes are co-cultured on opposite sides of the fiber wall with human brain microvascular endothelial cells (hBMECs) on the luminal side and astrocytes on the outside. The relatively thick fiber walls with small cylindrical pores limit communication between hBMECs and astrocytes.
The hollow fiber scaffold approach for artificial brain capillaries has significant advantages over 2D planar membranes. The cylindrical geometry in the hollow fiber allows for axial flow and shear stresses that are physiologically relevant. The ability to co-culture brain capillary endothelial cells and astrocytes on these structures has resulted in in vitro models exhibiting many of the important characteristics of the BBB. While this co-culture model exhibits some of the important characteristics of the BBB and represents a major technical accomplishment, at least several aspects can be improved: (1) the limited porosity of the polymer membrane restricts access of nutrients to the basal surfaces of the cells; (2) the large wall thickness (150 μm) and small pore diameter (0.5 μm) of the hollow fiber membrane limits contact between the hBMECs and astrocytes and severely restricts paracrine signaling; (3) the polymer surfaces are not tailored for BMEC and astrocyte culture, lacking appropriate architecture and cell adhesion ligands; (4) the polymer tubes themselves are much larger in diameter (330 μm inner diameter) than typical brain microvasculature; (5) the vessel is not embedded in an extracellular matrix; and (6) the platform does not allow direct imaging.